**2. Evidence of influenza virus infections in** *Arctocephalus australis* **individuals**

Marine mammals are susceptible to a variety of pathogens including influenza viruses. In humans, influenza causes annual epidemics and occasional pandemic diseases, with a significant threat to human health. In wild animals, several outbreaks have been reported and especially marine mammals experienced several devastating episodes that highlight the importance of monitoring wild populations to perform conservation programs and to evaluate possible risks to human health.

Influenza viruses belong to the *Orthomyxoviridae* family and are enveloped viruses with a segmented, negative-sense RNA genome (Webster et al., 1992). Embedded in the lipid envelope, the hemagglutinin (HA) and neuraminidase (NA) proteins are responsible for virus attachment and release from host cells, respectively (Webster et al., 1992). This family of viruses is composed of four genera: influenza A, B and C viruses and Thogoviruses (Wright & Webster, 2001). While influenza B and C viruses are primarily "human" viruses, influenza A viruses infect a variety of avian and mammalian species including humans, horses, swine and marine mammals such as seals and cetaceans (Wright & Webster, 2001; Webster et al., 1992). Influenza B virus was isolated from a harbor seal (*Phoca vitulina*) for the first time in the year 2000 (Osterhaus et al., 2000) becoming a possible second reservoir of this virus.

Influenza viruses are unique among respiratory tract viruses as they undergo considerable antigenic variation. Both surface antigens of the influenza A viruses are subject to two types of variation: drift and shift. Antigenic drift involves minor changes in the hemagglutinin (HA) and neuraminidase (NA) and plays a role in influenza epidemics, which occur sporadically. Antigenic shifts involve major changes in these molecules resulting from

replacement of the gene segment, producing new pandemic strains (Wright & Webster, 2001).

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 161

Indirect evidence of influenza A viruses has been reported in 27% of the South American fur seals sampled in Uruguay (Blanc et al., 2009). By Hemagglutination Inhibition Assay (HAI) it was found that all the positive samples reacted with A/New Caledonia/20/99(H1N1) antigen reaching HAI titer of 320 but none of the sampled serum reacted with A/Panamá/2007/99(H3N2) antigen. For the first time, the presence of influenza A in *A.* 

Influenza viruses have also been detected in whales. An H1N3 virus was isolated from a striped whale in the South Pacific (Lvov et al., 1978). In 1984 influenza A viruses of the H13N2 and H13N9 subtypes were isolated from a pilot whale (*Globicephala melas*) (Hinshaw et al., 1986). Serological, molecular, and biological analyses indicate that the whale isolates are closely related to the H13 influenza viruses from gulls (Hinshaw et al., 1986). In cetaceans, specific antibodies were observed in a low portion of sera from belugas

Few studies have been reported regarding the detection of influenza B viruses in marine mammals. The first one reported the isolation of influenza B virus (B/seal/Netherlands/1/99) from a naturally infected harbor seal in the year 2000. Sequence analyses as well as serology indicated that this influenza B virus is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies of the influenza B virus in 2% of the animals after 1995, and in none before that year, suggesting that the virus was introduced in the seal population from a human source around 1995 (Osterhaus et al., 2000). Antibodies to influenza B viruses were detected by ELISA in 14% and 10% of serum samples collected from Caspian seals in 1997

Serologic evidence of influenza B virus has been reported from South American Uruguayan fur seals *A. australis* (Blanc et al., 2009). Thirty of the 37 serum samples assayed by HAI reacted against one of the three antigens used: 25/37 (68%) reacted against B/Beijing/184/93 like viruses, 20/37 (54%) reacted against B/Hong Kong/330/01, and 24/37 (65%) reacted against B/Sichuan/379/99. The results show that 17 sera reacted against all B antigens, only six reacted against two antigens and eight sera did not react against any of them. The highest titer reached was (640) against B/sichuan antigen. The results demonstrated influenza B virus circulation in South American fur seals for the first time in our country and in this species. The antigens assayed correspond to strains that circulated in humans between the years 1999 and 2001, 3 to 5 years after the study was carried out, confirming the hypothesis of other authors that marine mammals could be a reservoir of influenza strains

It is important to consider that marine mammals share their habitat with several different wild shorebirds as well as with aquatic birds, the main influenza A virus hosts. The presence of bird feces in water, which can shed high concentrations of Avian Influenza viruses, and the close contact during feeding activities between birds and seals, increase the

*australis* was confirmed (Blanc et al., 2009) (Fig. 6).

and 2000, respectively (Ohishi et al., 2002).

that circulated in the past (Fig. 6).

probability of fecal-oral transmission.

(*Delphinapterus leucas*) in Arctic Canada (Nielsen et al., 2001).

Phylogenetic evidence suggests that influenza epidemics in humans and other mammals, including seals, come from mutation and antigenic drift of viruses originating from aquatic birds (Webster et al., 1992). Several influenza events which have affected marine mammals have been described since the late seventies. The New England coast was the scene of an episode of influenza virus between December 1979 and November 1980. More than 400 harbor seals (*Phoca vitulina*) died of acute pneumonia associated with the influenza virus A/Seal/Massachusetts/1/80 (H7N7). This was the first evidence of an influenza virus antigenically and genetically related to avian viruses that could be associated with severe disease in wild animals (Geraci et al., 1982; Lang et al., 1981; Webster et al., 1981). This H7N7 strain was associated with an approximate 20% mortality of the seal population and also showed potential for causing conjunctivitis in humans. However, it was not transmitted among humans.

A new event was described along the New England coast from June 1982 through March 1983. This time the influenza virus isolation was an H4N5 subtype, which had previously been detected only in birds. It was recovered from harbor seals dying of viral pneumonia (Hinshaw et al., 1984). This strain, which caused an estimated mortality of 2 % to 4 %, was found to be genetically and serologically related to avian strains.

In January 1991 and January to February 1992, influenza A viruses were isolated from seals that died of pneumonia along the Cape Cod Peninsula in Massachusetts. Antigenic characterization identified two H4N6 and three H3N3 viruses. This was the first isolation of an H3 influenza virus from seals, although this subtype is frequently detected in birds, pigs, horses and humans (Callan et al., 1995). Genetic analysis indicated that the viruses were both of avian origin and that transmission from birds to seals was the most likely possibility.

Also, indirect evidence of influenza infection was reported from a variety of marine mammal species. In pinnipeds, antibodies against influenza A virus were detected in sera from harp seals (*Phoca groenlandica*) and hooded seals (*Cystophora cristata*) collected between 1991 and 1992 in the Barents Sea (Steuen et al., 1994), as well as from sea lions (Otariidae) and seals in the North and Bering seas (De Boer et al., 1990), and a ringed seal (*Pusa hispida*) in Alaska (Danner et al., 1998). A serological survey of influenza A antibodies from five species of marine mammals collected from Arctic Canada between 1984 and 1998, revealed that 2.5% of ringed seals (*Phoca hispida*) were serologically positive (Nielsen et al., 2001). A serological study of influenza virus infection in Caspian seals (*Phoca caspica*) detected antibodies to human-related (H3N2) virus in 36% of the seals (Ohishi et al., 2002). Two years later, another study suggested that human-related H3 viruses were prevalent in Baikal seals *(Phoca sibirica*) and ringed seals (*Pusa hispida*) inhabiting the central Russian Arctic (Ohishi et al., 2004). Serological evidence of influenza A virus infection was reported in Kuril harbor seals (*Phoca vitulina stejnegeri*) of Hokkaido, Japan, from samples collected between 1998 and 2005 (Fujii et al., 2007). In this study, antibodies to H3 and H6 subtypes of influenza A virus were detected. This was the first time that H6 antibodies were identified in seals (Fujii et al., 2007).

Indirect evidence of influenza A viruses has been reported in 27% of the South American fur seals sampled in Uruguay (Blanc et al., 2009). By Hemagglutination Inhibition Assay (HAI) it was found that all the positive samples reacted with A/New Caledonia/20/99(H1N1) antigen reaching HAI titer of 320 but none of the sampled serum reacted with A/Panamá/2007/99(H3N2) antigen. For the first time, the presence of influenza A in *A. australis* was confirmed (Blanc et al., 2009) (Fig. 6).

160 New Approaches to the Study of Marine Mammals

2001).

among humans.

2007).

replacement of the gene segment, producing new pandemic strains (Wright & Webster,

Phylogenetic evidence suggests that influenza epidemics in humans and other mammals, including seals, come from mutation and antigenic drift of viruses originating from aquatic birds (Webster et al., 1992). Several influenza events which have affected marine mammals have been described since the late seventies. The New England coast was the scene of an episode of influenza virus between December 1979 and November 1980. More than 400 harbor seals (*Phoca vitulina*) died of acute pneumonia associated with the influenza virus A/Seal/Massachusetts/1/80 (H7N7). This was the first evidence of an influenza virus antigenically and genetically related to avian viruses that could be associated with severe disease in wild animals (Geraci et al., 1982; Lang et al., 1981; Webster et al., 1981). This H7N7 strain was associated with an approximate 20% mortality of the seal population and also showed potential for causing conjunctivitis in humans. However, it was not transmitted

A new event was described along the New England coast from June 1982 through March 1983. This time the influenza virus isolation was an H4N5 subtype, which had previously been detected only in birds. It was recovered from harbor seals dying of viral pneumonia (Hinshaw et al., 1984). This strain, which caused an estimated mortality of 2 % to 4 %, was

In January 1991 and January to February 1992, influenza A viruses were isolated from seals that died of pneumonia along the Cape Cod Peninsula in Massachusetts. Antigenic characterization identified two H4N6 and three H3N3 viruses. This was the first isolation of an H3 influenza virus from seals, although this subtype is frequently detected in birds, pigs, horses and humans (Callan et al., 1995). Genetic analysis indicated that the viruses were both of avian origin and that transmission from birds to seals was the most likely possibility. Also, indirect evidence of influenza infection was reported from a variety of marine mammal species. In pinnipeds, antibodies against influenza A virus were detected in sera from harp seals (*Phoca groenlandica*) and hooded seals (*Cystophora cristata*) collected between 1991 and 1992 in the Barents Sea (Steuen et al., 1994), as well as from sea lions (Otariidae) and seals in the North and Bering seas (De Boer et al., 1990), and a ringed seal (*Pusa hispida*) in Alaska (Danner et al., 1998). A serological survey of influenza A antibodies from five species of marine mammals collected from Arctic Canada between 1984 and 1998, revealed that 2.5% of ringed seals (*Phoca hispida*) were serologically positive (Nielsen et al., 2001). A serological study of influenza virus infection in Caspian seals (*Phoca caspica*) detected antibodies to human-related (H3N2) virus in 36% of the seals (Ohishi et al., 2002). Two years later, another study suggested that human-related H3 viruses were prevalent in Baikal seals *(Phoca sibirica*) and ringed seals (*Pusa hispida*) inhabiting the central Russian Arctic (Ohishi et al., 2004). Serological evidence of influenza A virus infection was reported in Kuril harbor seals (*Phoca vitulina stejnegeri*) of Hokkaido, Japan, from samples collected between 1998 and 2005 (Fujii et al., 2007). In this study, antibodies to H3 and H6 subtypes of influenza A virus were detected. This was the first time that H6 antibodies were identified in seals (Fujii et al.,

found to be genetically and serologically related to avian strains.

Influenza viruses have also been detected in whales. An H1N3 virus was isolated from a striped whale in the South Pacific (Lvov et al., 1978). In 1984 influenza A viruses of the H13N2 and H13N9 subtypes were isolated from a pilot whale (*Globicephala melas*) (Hinshaw et al., 1986). Serological, molecular, and biological analyses indicate that the whale isolates are closely related to the H13 influenza viruses from gulls (Hinshaw et al., 1986). In cetaceans, specific antibodies were observed in a low portion of sera from belugas (*Delphinapterus leucas*) in Arctic Canada (Nielsen et al., 2001).

Few studies have been reported regarding the detection of influenza B viruses in marine mammals. The first one reported the isolation of influenza B virus (B/seal/Netherlands/1/99) from a naturally infected harbor seal in the year 2000. Sequence analyses as well as serology indicated that this influenza B virus is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies of the influenza B virus in 2% of the animals after 1995, and in none before that year, suggesting that the virus was introduced in the seal population from a human source around 1995 (Osterhaus et al., 2000). Antibodies to influenza B viruses were detected by ELISA in 14% and 10% of serum samples collected from Caspian seals in 1997 and 2000, respectively (Ohishi et al., 2002).

Serologic evidence of influenza B virus has been reported from South American Uruguayan fur seals *A. australis* (Blanc et al., 2009). Thirty of the 37 serum samples assayed by HAI reacted against one of the three antigens used: 25/37 (68%) reacted against B/Beijing/184/93 like viruses, 20/37 (54%) reacted against B/Hong Kong/330/01, and 24/37 (65%) reacted against B/Sichuan/379/99. The results show that 17 sera reacted against all B antigens, only six reacted against two antigens and eight sera did not react against any of them. The highest titer reached was (640) against B/sichuan antigen. The results demonstrated influenza B virus circulation in South American fur seals for the first time in our country and in this species. The antigens assayed correspond to strains that circulated in humans between the years 1999 and 2001, 3 to 5 years after the study was carried out, confirming the hypothesis of other authors that marine mammals could be a reservoir of influenza strains that circulated in the past (Fig. 6).

It is important to consider that marine mammals share their habitat with several different wild shorebirds as well as with aquatic birds, the main influenza A virus hosts. The presence of bird feces in water, which can shed high concentrations of Avian Influenza viruses, and the close contact during feeding activities between birds and seals, increase the probability of fecal-oral transmission.

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 163

In Uruguay, the first isolation of *Mycobacterium* spp. in pinnipeds was conducted in 1987 from samples of South American sea lion *O. flavescens* specimens kept in "Villa Dolores" municipal zoo. Of the ten animals studied, one died and nine others were tuberculinpositive and were later euthanized. Seven animals showed typical histo-pathological lesions of tuberculosis, and a total of 6 strains were isolated. Initially, it was considered that the bacillus was *M. bovis* according to results from a smear, biochemical tests and culture features. The strains were inoculated to guinea pigs (0,1 mg) that developed characteristic lesions and subsequently *Mycobacterium* spp. were isolated, fulfilling Koch's postulates (Castro-Ramos et al., 1998). In 1997 *Mycobacterium* spp. was isolated from lung samples of an adult South American fur seal stranded on the coast of Montevideo. The animal was collected from the beach by a NGO and sent to quarantine in a zoo for recovery, but died four days after admission. The observed granulomatous lung lesions were typical of TB, and

Between 2001 and 2006, pathological, microbiological and genetic studies were conducted on dead stranded animals of different species of pinnipeds found along ocean shores of Uruguay: South American fur seal (n = 129), South American sea lion (n = 24) and Southern elephant seal (n = 1). Necropsies were performed using standard methods (Dierauf, 1990). Samples from several organs with or without lesions (lung, mediastinal lymph nodes,

Formalin fixed samples were processed by standard histological methods: 4-5 cuts were made at 5-6 µm and stained with Hematoxylin-Eosin and Ziehl-Neelsen (ZN) (Luna, 1968). Mycobacteriological studies were performed according to the methodology described by the Pan American Zoonoses Center (Centro Panamericano de Zoonosis (OPS/OMS) (1979), Office International des Epizooties (OIE) (2000), Runyon et al., (1980) and Tacquet et al., (1967). Smears from single or pooled samples of each animal were performed and then cultured in Stonebrink and Lowenstein Jensen media. Cultures were kept for eight weeks at 37° C and periodically reviewed. Culture tests were based on microscopic features, morphology of the colony, growth temperature, time of development and cromogenicity of isolates. The identification was completed with the following biochemical tests: niacin, nitrate reduction, catalase at 22° C and 68° C, hydrolysis of Tween 80 at 5 and 10 days, reduction of potassium tellurite 0.2% at 3 days, urease and pyrazinamidase. A total of 14

Strains isolated in 1987 (N° 01073, adult male *O. flavescens*), 1997 (N ° 01337, juvenile male *A. australis*) and 2002 (N ° 2493, juvenile female *O. flavescens*) were analyzed through amplification of 200 bp of the Internal Transcribed Spacer (ITS) region through Polymerase Chain Reaction (PCR) as in Roth et al., (2000). Sequences obtained were compared to those available at GenBank database through a maximum parsimony phylogenetic tree, and

During the necropsies, granulomatous lesions were observed in only five animals: two South American sea lions (juvenile male and female) and three South American fur seals (two adult and one juvenile male) (Fig. 7), from which *M. tuberculosis / M. pinnipedii* complex

strains were grouped with sequences of *M. tuberculosis / M. pinnipedii*.

spleen, liver) (n = 36) were stored at 4° C, frozen at -20° C or fixed in 10% formalin.

a *Mycobacterium* spp. strain was isolated (Castro-Ramos et al., 2001).

strains were isolated (Table 1).

**Figure 6.** Antibodies to Influenza A and B virus by HAI in fur seal sera. Percent (number positive/number tested) of samples bearing antibodies vs. HAI titer for each Influenza antigen assayed. Titers ≥ 80 were considered positive. Antigens used: A/New Caledonia/20/99(H1N1), A/Panamá/2007/99(H3N2), B/Beijing/184/93-like viruses, B/Yamanashi/166/98, B/Hong Kong/330/01, and B/Sichuan/379/99.

Monitoring the distribution of the influenza virus in wild animal species including marine mammals is important for understanding the ecology and evolution of the virus, and also to understand how the virus can mutate and re-emerge more virulent, producing devastating epidemic diseases.
